绵羊胚胎FGF5基因单碱基突变体系的建立

doi:10.16431/j.cnki.1671-7236.2021.10.004

Abstract

Abstract: The purpose of this study was to modify the exon 1 of sheep (Ovis aries) fibroblast growth factor 5 (FGF5) gene by single base editing technique to introduce the stop codon, to obtain sheep embryos with site-directed editing, and to provide experimental materials for breeding sheep with long wool traits. Firstly, four single guide RNA (sgRNA) oligonucleotide chains (sgRNA-T1 to sgRNA-T4) were designed and synthesized, and four groups of recombinant expression vectors were constructed. The constructed pGL3-U6-sgRNA-PGK-puromycin and pCMV-AncBE4max-P2A-GFP plasmids were transferred into four groups of sheep fibroblasts by co-transfection, and then the activity of the transfected cells was detected by Cruiser^TM enzyme and sequenced at embryos level. The results showed that the PCR products of sgRNA-T1 and sgRNA-T4 groups with targeted effects were screened at cell level and could be cleaved by Cruiser^TM enzyme, and the sequencing results showed that both groups had targeted effects, with editing efficiency of 68.75% and 47.37%, respectively. Different concentrations of AncBE4max mRNA and effective sgRNA were mixed and transforred into sheep parthenogenetic activated embryos by microinjection technique, and the cleavage rate, blastocyst rate and editing efficiency of the embryos were detected. The results showed that at the embryo level, the best injection concentration combination of AncBE4max(ng/μL):sgRNA(ng/μL) was 100:50. The sequencing results of single embryos randomly selected from this concentration group showed that the editing efficiency of introducing stop codons was 80%. However, no editing was detected in the injected embryos with different concentrations of sgRNA-T1. In this study, aiming at the exon 1 of FGF5 gene, two sgRNA (T1, T4) targeting sheep FGF5 gene were successfully screened by transfection expression vector in fibroblasts, and the transformation of the target site C→T of exon 1 of FGF5 gene was successfully realized in embryos by microinjection of sheep parthenogenetic activated embryos, which laid a foundation for the production of sheep with FGF5 gene targeted editing in the later stage.

Key words: sheep; fibroblast growth factor 5 (FGF5); single base editing

CLC Number:

S826

MENG Yaqi, YAO Xudong, REN Xiumeiao, GUO Yanhua, TANG Hong, ZHANG Yiyuan, WANG Limin, ZHOU Ping. Establishment of Single Base Mutation System of FGF5 Gene in Sheep Embryos[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3533-3544.

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Establishment of Single Base Mutation System of FGF5 Gene in Sheep Embryos

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